Isolated nona-and decapeptides which bind to HLA molecules, and the use thereof

ABSTRACT

Nonapeptides and decapeptides which bind to HLA molecules and provoke proliferation of cytolytic T cells are disclosed. The decapeptides terminate in Valine, and are restricted in their first three amino acid positions. Other useful nonapeptides are also disclosed.

RELATED APPLICATION

[0001] This is a continuation-in-part of Ser. No. 09/061,388, filed Jan.13, 1998, which is a continuation in part of Ser. No. 08/880,963, filedJun. 23, 1997, incorporated by reference. A portion of the invention waspublished by the inventors, less than one year before the filing date ofthe first continuation-in-part application. See Romero et al., J.Immunol. 159: 2366 (1997) incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to peptides which are useful in thecontext of cellular immunology. More particularly, the invention relatesto peptides which bind to HLA molecules on the surface of cells. Atleast some of these peptides also induce the activation of cytolytic Tcells, when they are complexed with their partner HLA molecule. Also apart of the invention are the uses of these peptides in areas such asidentifying HLA-A2 positive cells, provoking T cells, determiningpresence of particular T cells, as well as cytolytic T cells themselves.

BACKGROUND AND PRIOR ART

[0003] The study of the recognition or lack of recognition of cancercells by a host organism has proceeded in many different directions.Understanding of the field presumes some understanding of both basicimmunology and oncology.

[0004] Early research on mouse tumors revealed that these displayedmolecules which led to rejection of tumor cells when transplanted intosyngeneic animals. These molecules are “recognized” by T-cells in therecipient animal, and provoke a cytolytic T-cell response with lysis ofthe transplanted cells. This evidence was first obtained with tumorsinduced in vitro by chemical carcinogens, such as methylcholanthrene.The antigens expressed by the tumors and which elicited the T-cellresponse were found to be different for each tumor. See Prehn, et al.,J. Natl. Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20:1561-1572 (1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio,Cancer Res. 30: 2458-2462 (1970) for general teachings on inducingtumors with chemical carcinogens and differences in cell surfaceantigens. This class of antigens has come to be known as “tumor specifictransplantation antigens” or “TSTAs.” Following the observation of thepresentation of such antigens when induced by chemical carcinogens,similar results were obtained when tumors were induced in vitro viaultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53: 333-1336(1974).

[0005] While T-cell mediated immune responses were observed for thetypes of tumor described supra, spontaneous tumors were thought to begenerally non-immunogenic. These were therefore believed not to presentantigens which provoked a response to the tumor in the tumor carryingsubject. See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).

[0006] The family of tum⁻ antigen presenting cell lines are immunogenicvariants obtained by mutagenesis of mouse tumor cells or cell lines, asdescribed by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), thedisclosure of which is incorporated by reference. To elaborate, tum⁻antigens are obtained by mutating tumor cells which do not generate animmune response in syngeneic mice and will form tumors (i.e., “tum⁺”cells). When these tum⁺ cells are mutagenized, they are rejected bysyngeneic mice, and fail to form tumors (thus “tum⁻”). See Boon et al.,Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which isincorporated by reference. Many tumor types have been shown to exhibitthis phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).

[0007] It appears that turn variants fail to form progressive tumorsbecause they initiate an immune rejection process. The evidence in favorof this hypothesis includes the ability of “tum⁻” variants of tumors,i.e., those which do not normally form tumors, to do so in mice withimmune systems suppressed by sublethal irradiation, Van Pel et al.,Proc. Natl. Acad. Sci. USA 76: 5282-5285 (1979); and the observationthat intraperitoneally injected tum⁻ cells of mastocytoma P815 multiplyexponentially for 12-15 days, and then are eliminated in only a few daysin the midst of an influx of lymphocytes and macrophages (Uyttenhove etal., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes theobservation that mice acquire an immune memory which permits them toresist subsequent challenge to the same tum⁻ variant, even whenimmunosuppressive amounts of radiation are administered with thefollowing challenge of cells (Boon et al., Proc. Natl. Acad. Sci. USA74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).Later research found that when spontaneous tumors were subjected tomutagenesis, immunogenic variants were produced which did generate aresponse. Indeed, these variants were able to elicit an immuneprotective response against the original tumor. See Van Pel et al., J.Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it ispossible to elicit presentation of a so-called “tumor rejection antigen”in a tumor which is a target for a syngeneic rejection response. Similarresults have been obtained when foreign genes have been transfected intospontaneous tumors. See Fearon et al., Cancer Res. 48: 2975-1980 (1988)in this regard.

[0008] A class of antigens has been recognized which are presented onthe surface of tumor cells and are recognized by cytolytic T cells,leading to lysis. This class of antigens will be referred to as “tumorrejection antigens” or “TRAs” hereafter. TRAs may or may not elicitantibody responses. The extent to which these antigens have beenstudied, has been via cytolytic T cell characterization studies, invitro, i.e., the study of the identification of the antigen by aparticular cytolytic T cell (“CTL” hereafter) subset. The subsetproliferates upon recognition of the presented tumor rejection antigen,and the cells presenting the tumor rejection antigens are lysed.Characterization studies have identified CTL clones which specificallylyse cells expressing the tumor rejection antigens. Examples of thiswork may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boonet al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol.124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634(1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladinoet al., Cancer. Res. 47: 5074-5079 (1987). This type of analysis isrequired for other types of antigens recognized by CTLs, including minorhistocompatibility antigens, the male specific H—Y antigens, and theclass of antigens referred to as “tum−” antigens, and discussed herein.

[0009] A tumor exemplary of the subject matter described supra is knownas P815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278(1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al.,J. Exp. Med. 172: 35-45 (1990), the disclosures of which areincorporated by reference. The P815 tumor is a mastocytoma, induced in aDBA/2 mouse with methylcholanthrene and cultured as both an in vitrotumor and a cell line. The P815 line has generated many tum⁻ variantsfollowing mutagenesis, including variants referred to as P91A (DePlaen,supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast totumor rejection antigens—and this is a key distinction—the tum⁻ antigensare only present after the tumor cells are mutagenized. Tumor rejectionantigens are present on cells of a given tumor without mutagenesis.Hence, with reference to the literature, a cell line can be tum⁺, suchas the line referred to as “P1,” and can be provoked to producetum-variants. Since the tum⁻ phenotype differs from that of the parentcell line, one expects a difference in the DNA of tum⁻ cell lines ascompared to their tum⁺ parental lines, and this difference can beexploited to locate the gene of interest in tum⁻ cells. As a result, itwas found that genes of tum⁻ variants such as P91A, 35B and P198 differfrom their normal alleles by point mutations in the coding regions ofthe gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58:293-303 (1989). This has proved not to be the case with the TRAs of thisinvention. These papers also demonstrated that peptides derived from thetum⁻ antigen are presented by H-2^(d) Class I molecules for recognitionby CTLs. P91A is presented by L^(d), P35 by D^(d) and P198 by K^(d).

[0010] PCT application PCT/US92/04354, filed on May 22, 1992 assigned tothe same assignee as the subject application, teaches a family of humantumor rejection antigen precursor coding genes, referred to as the MAGEfamily. Several of these genes are also discussed in van der Bruggen etal., Science 254: 1643 (1991). It is now clear that the various genes ofthe MAGE family are expressed in tumor cells, and can serve as markersfor the diagnosis of such tumors, as well as for other purposesdiscussed therein. See also Traversari et al., Immunogenetics 35: 145(1992); van der Bruggen et al., Science 254: 1643 (1991) and De Plaen,et al., Immunopenetics 40: 360 (1994). The mechanism by which a proteinis processed and presented on a cell surface has now been fairly welldocumented. A cursory review of the development of the field may befound in Barinaga, “Getting Some ‘Backbone’: How MHC Binds Peptides,”Science 257: 880 (1992); also, see Fremont et al., Science 257: 919(1992); Matsumura et al., Science 257: 927 (1992); Engelhard, Ann. Rev.Immunol. 12:181-207 (1994); Madden, et al, Cell 75:693-708 (1993);Ramensee, et al, Ann. Rev. Immunol. 11:213-244 (1993); Germain, Cell 76:287-299 (1994). These papers generally point to a requirement that thepeptide which binds to an MHC/HLA molecule be nine amino acids long (a“nonapeptide”), and to the importance of the second and ninth residuesof the nonapeptide.. For H-2K^(b), the anchor residues are positions 5and 8 of an octamer, for H-2D^(b), they are positions 5 and 9 of anonapeptide while the anchor residues for HLA-A1 are positions 3 and 9of a nonamer. Generally, for HLA molecules, positions 2 and 9 areanchors.

[0011] Studies on the MAGE family of genes have now revealed that aparticular nonapeptide is in fact presented on the surface of some tumorcells, and that the presentation of the nonapeptide requires that thepresenting molecule be HLA-A1. Complexes of the MAGE-1 tumor rejectionantigen (the “TRA” or “nonapeptide”) leads to lysis of the cellpresenting it by cytolytic T cells (“CTLs”).

[0012] Research presented in, e.g., U.S. Pat. No. 5,405,940 filed Aug.31, 1992, and in U.S. Pat. No.5,571,711, found that when comparinghomologous regions of various MAGE genes to the region of the MAGE-1gene coding for the relevant nonapeptide, there is a great deal ofhomology. Indeed, these observations lead to one of the aspects of theinvention disclosed and claimed therein, which is a family ofnonapeptides all of which have the same N-terminal and C-terminal aminoacids. These nonapeptides were described as being useful for variouspurposes which includes their use as immunogens, either alone or coupledto carrier peptides. Nonapeptides are of sufficient size to constitutean antigenic epitope, and the antibodies generated thereto weredescribed as being useful for identifying the nonapeptide, either as itexists alone, or as part of a larger polypeptide.

[0013] The preceding survey of the relevant literature shows thatvarious peptides, usually eight, nine, or ten amino acids in length,complex with MHC molecules and present targets for recognition bycytolytic T cells. A great deal of study has been carried out onmelanoma, and melanoma antigens which are recognized by cytolytic Tcells are now divided into three broad categories. The first, whichincludes many of the antigens discussed, supra, (e.g., MAGE), areexpressed in some melanomas, as well as other tumor types, and normaltestis and placenta. The antigens are the expression product of normalgenes which are usually silent in normal tissues.

[0014] A second family of melanoma antigens includes antigens which arederived from mutant forms of normal proteins. Examples of this familyare MUM-1 (Coulie, et al, Proc. Natl. Acad. Sci. USA 92:7976-7980(1955)); CDK4 (Wölfel, et al, Science 269:1281-1284(1955)); Bcatenin(Robbins, et al, J. Exp. Med. 183:1185-1192 (1996)); and HLA-A2(Brandel, et al, J. Exp. Med. 183:2501-2508 (1996)). A third category,also discussed, supra, includes the differentiation antigens which areexpressed by both melanoma and melanocytes. Exemplary are tyrosinase,gp100, gp75, and Melan A/Mart-1. See U.S. Pat. No.5,620,886 incorporatedby reference, with respect to Melan-A. See Wölfel, et al., Eur. J.Immunol. 24: 759 (1994) and Brichard, et al., Eur. J. Immunol. 26: 224(1996) for tyrosinase; Kang, et al., J. Immunol. 155: 1343 (1995); Cox,et al., Science 264: 716 (1994); Kawakami, et al., J. Immunol. 154: 3961(1995) for gp 100; Wang, et al., J. Exp. Med. 183: 1131 (1996) for gp75.

[0015] Cytolytic T cells (“CTLs” hereafter) have been identified inperipheral blood lymphocytes, and tumor infiltrating lymphocytes, ofmelanoma patients who are HLA-A^(·)0201 positive. See Kawakami, et al,Proc. Natl. Acad. Sci. USA 91:3515 (1994); Coulie, et al, J. Exp. Med.180:35 (1994). When ten HLA-A^(·)0201 restricted Melan-A specific CTLsderived from different patients were tested, nine of them -were found torecognize and react with the peptide Ala Ala Gly Ile Gly Ile Leu ThrVal, (SEQ ID NO: 2), which consists of amino acids 27-35 of Melan-A.(Kawakami, et al, J. Exp. Med. 180:347-352 (1994)). Rivoltini, et al, J.Immunol. 154:2257 (1995), showed that Melan-A specific CTLs could beinduced by stimulating PBLs from HLA-A^(·)0201 positive normal donors,and melanoma patients, using SEQ ID NO: 2. The strength of this responsehas led to SEQ ID NO: 2 being proposed as a target for vaccinedevelopment. It has now been found, however, that a decapeptide, i.e.,

Glu Ala Ala Gly Ile Gly Ile Leu Thr Val

[0016] (SEQ ID NO: 1), is actually a better target than SEQ ID NO: 2.This recognition has led to work set forth herein, which is part of theinvention.

[0017] The majority of peptides which have been identified as binding toHLA-A^(·)0201 are 9 or 10 amino acids in length, and are characterizedby two anchor residues. The first is Leu or Met at position 2, and thesecond is Leu or Val at position 9. See Falk, et al, Nature 351:290(1991). Ruppert, et al, in Cell 74:929 (1993), show that amino acidsfound at other positions within a nona-or decapeptide may also have arole in the peptide-HLA-A^(·)0201 interaction. They show, e.g., that anegatively charged residue or proline at position 1 was associated withpoor HLA-A^(·)0201 binding.

[0018] What is interesting about this work is that the two peptidesrepresented by SEQ ID NOS: 1 and 2 do not possess the major anchorresidue at position 2 and, the strong binder SEQ ID NO: I has anegatively charged residue at position 1.

[0019] A strong binder is not necessarily a stable binder, meaning thatthe interaction between peptide and HLA molecule may be, and is, brief.When it is desired to induce CTLs, to identify them or to carry outother types of experiments, it would be desirable to have a peptide withthe ability to bind to an MHC Class I molecule which binds with highaffinity and forms stable complexes. See Van der Burg et al., J.Immunol, 156:3308 (1996).

[0020] The invention involves, inter alia, the development of newnonamers and decamers which are surprisingly good HLA binders and CTLstimulators. These molecules, as well as their uses, are among thefeatures of the invention which are set forth in the disclosure whichfollows.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIG. 1 shows results of experiments carried out to determine iftumor infiltrating lymphocyte populations would lyse cells presentingcomplexes of HLA-A^(·)0201 and various peptides on their surfaces.

[0022]FIGS. 2a-2 d show stability studies comparing various peptides.

[0023]FIG. 3 shows antigenic activity of various peptides when testedwith TILNs.

[0024]FIG. 4 parallels FIG. 3, but uses CTLs generated from PBLs bystimulation with various peptides.

[0025]FIGS. 5a-5 r show results of flow cytometry studies, followingstimulation of PBMCs with various peptides.

[0026]FIGS. 6a-6 e depict results of lytic activity assays on PBMCswhich have been stimulated with various peptides.

[0027]FIGS. 7a-7 e present data on Melan-A specific lytic activity offluorescently sorted lymphocytes positive for tetramers containing SEQID NO: 1, following stimulation with other peptides.

[0028]FIG. 8 shows quantitative assessment of peptide dependent lyticactivity of a Melan-A monospecific CTL line.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0029] In these experiments tumor infiltrating lymphocytes (“TILs”hereafter), were generated from tumor invaded lymph nodes of patientswho were HLA-A^(·)0201 positive. The experiments were designed so as toavoid antigen specific selection in vitro, and the methodology is nowset forth.

[0030] Biopsies of tumor infiltrated lymph nodes (“TILNs” hereafter)were lysed to single cell suspensions, and then cultured in 24 wellculture plates. A total of 3×10⁶ cells were added to 2 mls of Iscove'sDulbecco medium which had been supplemented with Asn (0.24 mM), Arg(0.55 mM), and Gln (1.5 mM), and 10% pooled human A⁺ serum (serumobtained from type A blood donors), together with recombinant human IL-2(100 u/ml), and IL-7 (10 ng/ml). These were the only cytokines used toculture the cell suspensions, so as to avoid antigen specific selectionin vitro. The suspensions were cultured for 2-3 weeks, and the cellswere then characterized for cell surface phenotype. Only populationswith greater than 75% CD8⁺T cells, and of desired cytolytic activitywere used. This second property was determined by combining the TILNpopulations with autologous cells, a melanoma cell line previouslycharacterized as being HLA-A^(·)0201 positive (Me290), a melanoma cellline known to be HLA-A*0201 negative (Me260) or cell line T2, which doesnot process antigen, together with the peptide of SEQ ID NO: 1. Thepeptide was added at 1 μM, together with varying ratios of effector(TILN) cells, and the target cells. The results presented in FIG. 1,show results obtained using LAU 132 and LAU 203, two TILN populationsidentified via this method. In FIG. 1, “M10” is SEQ ID NO: 1, and theadditional abbreviations are as set forth, supra. The assay was a 4-hour⁵¹Cr release assay, carried out in the absence or presence ofexogenously added peptide. In FIG. 1, open symbols stand for the absenceof the peptide, and solid symbols for its presence. In this assay, thetarget cells were labelled with ⁵¹Cr for one hour, at 37° C., and werethen washed two times. Labeled cells (1000 cells in 50 μl) were added toa 50 μl sample of effector cells (varying amounts, as indicated herein),in the presence or absence of 50 μl of antigenic peptide (1 μg/ml).Prior to their addition, the effector cells had been incubated for aminimum of 20 minutes at 37° C., in the presence of unlabelled naturalkiller (NK) cell targets (50,000 cells per well), in order to eliminateany non-specific lysis due to NK-like effectors present in the effectorpopulation. The ⁵¹Cr release was measured after 4 hours of incubation at37° C., and percent specific lysis was calculated as:$100\left\lbrack \frac{\left( {{{experimental}\quad {release}} - {{spontaneous}\quad {release}}} \right)}{{{total}\quad {release}} - {{spontaneous}\quad {release}}} \right\rbrack$

[0031] As FIG. 1 shows, the two TILN populations lysed the HLA-A^(·)0201positive cell line equally well, whether or not the peptide was added.The HLA-A*0201 negative line, Me260, was not lysed in either situation,and T2, which does not process antigen, was lysed only when the peptidewas added. These results show that the two TILN populations usedhereafter recognize the epitope defined by SEQ ID NO: 1, when complexedto HLA-A^(·)0201 positive cells.

EXAMPLE 2

[0032] The experiments described, supra, were modified somewhat, todetermine if the TILNs recognized other peptides better than SEQ IDNO: 1. In these experiments, the following peptides were synthesized,using known methods: Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 2)Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 3) Ile Ile Leu Thr ValIle Leu Gly Val Leu (SEQ ID NO: 4)

[0033] These peptides correspond, respectively, to amino acids 27-35,27-36, and 32-40 of Melan-A.

[0034] TILN recognition was determined by incubating T2 cells (target),with TILNs (effector), at an effector: target ratio of 30:1. Varyingconcentrations of the peptides SEQ ID NOS: 1, 2, 3, or 4 were used. The⁵¹Cr release assay discussed, supra, was used. The following Table setsforth the results of these experiments, wherein the peptideconcentration is that which gave 50% of maximum activity. Relativeactivity is that obtained via comparison to SEQ ID NO: 2, i.e.,: [nM]50%[SEQ ID NO: 2]/[nM]50% [test peptide]. TABLE I TILN LAU 203 TILN LAU 132Peptide Peptide^(a) Relative Peptide Relative Sequence [nM] 50%activity^(b) [nM] 50% activity AAGIGIL 40 1 15 1 TV₂₇₋₃₅ EAAGIGIL 1.5 271 15 TV₂₆₋₃₅ AAGIGIL 600 0.06 300 0.05 TVI₂₇₋₃₆ ILTVILG >10⁴ <4 × 10⁻³>10⁴ <1.5 × 10⁻³ V₃₂₋₄₀

[0035] It will be seen that SEQ ID NO: 1 had significantly higheractivity than the other peptides tested.

EXAMPLE 3

[0036] A series of peptides were then synthesized, in order to attemptto determine peptides with enhanced binding to HLA-A^(·)0201 molecules.The peptides synthesized are considered to be derivatives of SEQ ID NO:2 (i.e., Ala Ala Gly Ile Gly Ile Leu Thr Val), and are Ala Leu Gly IleGly Ile Leu Thr Val (SEQ ID NO: 5) Ala Met Gly Ile Gly Ile Leu Thr Val(SEQ ID NO: 6) Leu Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 7) andMet Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 8)

[0037] For SEQ ID NO: 1, i.e., Glu Ala Ala Gly Ile Gly Ile Leu Thr Valthe derivatives were: Glu Leu Ala Gly Ile Gly Ile Leu Thr Val (SEQ IDNO: 9) Glu Met Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 10) Glu AlaLeu Gly Ile Gly Ile Leu Thr Val (SEQ ED NO: 11) Glu Ala Met Gly Ile GlyIle Leu Thr Val (SEQ ID NO: 12) Tyr Ala Ala Gly Ile Gly Ile Leu Thr Val(SEQ ID NO: 13) Phe Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 14)Ala Ala Ala Gly Ile Gly Ile Leu Thr Val (SEQ ID NO: 15) and Ala Leu AlaGly Ile Gly Ile Leu Thr Val (SEQ ID NO: 16)

[0038] Three other control peptides were used, i.e.: Glu Ile Leu Gly PheVal Phe (SEQ ID NO: 17) Thr Leu Glu Val Asp Pro Ile Gly His (SEQ ID NO:18) Leu Tyr and Phe Leu Trp Gly Pro Arg Ala (SEQ ID NO: 19) Leu Val.

[0039] SEQ ID NOS: 17 and 18 correspond to amino acids 58-66 ofInfluenza A matrix protein (“FLUMA”), and amino acids 168-176 of MAGE-3TRAP.

[0040] In these experiments a peptide known to bind to HLA-A*0201 wasused, i.e., amino acids 271-279 of the MAGE-3 TRAP (SEQ ID NO: 19), inan inhibition assay, together with cytolytic T cell line 198NS (Valmori,et al., Canc. Res. 57:735 (1997) which recognizes complexes of SEQ IDNO: 19 and HLA-A*0201. In these assays, varying concentrations of testpeptides (1 μM to 100 μM) were incubated with ⁵¹Cr labelled T2 cells(1000 cells/well), for 15 minutes, at room temperature. A suboptimaldose of SEQ ID NO: 19 was then added (1 nM), together with CTL 198NS, inan amount sufficient to create a 5/1 effector/target ratio. A ⁵¹Crrelease assay was then carried out, in accordance with the method setforth, supra. The amount of test peptide needed to inhibit recognitionof complexes by the CTL was calculated, and then binding affinity ofeach peptide, relative to SEQ ID NO: 2, was calculated, using theformula:$R = \frac{{ID}_{50}\left( {{SEQ}\quad {ID}\quad {NO}\text{:}2} \right)}{{ID}_{50}\left( {{test}\quad {peptide}} \right)}$

[0041] If R is greater than one, then the tested peptide bound toHLA-A*0201 with greater affinity than SEQ ID NO: 2. A value less thanone indicates lower affinity. The results are set forth below: TABLE IIRelative Competitor² competitor Peptide Sequence [μM] 50% activity^(b)Melan-A₂₇₋₃₅ AAGIGILTV 60 1 ALGIGILTV 1.5 40 AMGIGILTV 2 30 LAGIGILTV 651 MAGIGILTV 55 1 Melan-A₂₆₋₃₅ EAAGIGILTV 15 4 ELAGIGILTV 6.5 9EMAGIGILTV 20 3 EALGIGILTV 100 0.6 EAMGIGILTV 100 0.6 YAAGIGILTV 4 15FAAGIGILTV 2 30 Influenza A matrix₅₈₋₆₆ GILGFVFTL 1 60 MAGE-3₁₆₈₋₁₇₆EVDPIGHLY >100 <0.6

[0042] SEQ ID NOS: 1, 5, 6, 9, 10, 13 and 14 all showed higher affinitythan SEQ ID NO:2.

EXAMPLE 4

[0043] One concern in developing MHC binding peptides is that theresulting complexes of MHC molecule and peptide be stable, preferablymore stable than the peptide originally found complexed to the MHCmolecule.

[0044] To test stability of the newly synthesized peptides, T2 cellswere incubated, overnight, at room temperature in serum free medium withsaturating amounts of peptide (10 uM), and 3 μg/ml β-microglobulin, tofacilitate the assembly of the requisite MHC molecules. Peptides werethen removed, and 10⁻⁴M ementine (which inhibits protein synthesis) wasadded. The cells were then incubated at 37° C. for varying periods oftime. Aliquots of cells were stained, at various points of theincubation, with a labelled HLA-A2 specific mAb to measure HLA-A2expression.

[0045] Stability was determined by comparison with complexes involvingSEQ ID NO: 17 which were stable over a 6-hour period. The results arepresented in FIGS. 2A-D. FIG. 2A shows mean fluorescence intensity foreach peptide. “NC” refers to HLA-A*0201, presented by T2 cells, in theabsence of exogenous peptide, while “FLUMA” is SEQ ID NO: 17, and is anacronym for “Flu matrix antigen.” In FIG. 2A, the peptides are SEQ IDNOS: 2, 1, and 9. In FIG. 2B, they are SEQ ID NOS: 2, 7, 8, 5, 6, and 17(“FLUMA”). In FIG. 2C, they are SEQ ID NOS: 1, 12, 11, 9, 10, and 17. InFIG. 2D, they are SEQ ID NOS: 1 13, 14, and 17. The breakup is solely tofacilitate the review. FIGS. 2B-2D show relative complex stability wherefluorescent intensities with test peptides were normalized, relative tothe stability observed when using SEQ ID NO: 17. SEQ ID NOS: 1 and 2both form unstable complexes, which decay within one hour. This was alsofound with SEQ ID NOS: 7 and 8.

[0046] On the other hand, SEQ ID NOS: 9, 10, 13, and 14 formed stablecomplexes over a 6-hour period, while SEQ ID NOS: 5, 6, 11, and 12formed complexes of intermediate stability.

EXAMPLE 5

[0047] The antigenic activity of each of the peptides presented, supra,when the peptide was associated with HLA-A*0201, was tested in a ⁵¹Crassay of the type discussed, supra, using TILNs, and CTLs. Dose responseanalyses were performed on each peptide, and antigenic activity,relative to SEQ ID NO: 2, was calculated. These values are set forth inthe following Tables III and IV and FIG. 3 which present data fromTILNs, (Table III and FIG. 3) and CTLs (Table IV), respectively.

[0048] Substituting the N-terminal amino acid of SEQ ID NO: 2 with Leuor Met enhanced activity between 7.5 and 20 fold, while substitutions atthe second position nearly abolished it, even though binding toHLA-A*−0201 was increased (Table III and FIG. 3).

[0049] SEQ ID NO: 1 was better recognized than SEQ ID NO: 2, andsubstitution of Ala in the second position of SEQ ID NO: 1 increasedrecognition 30- and 600 fold, respectively. Such substitutions atposition 3 reduced activity, which was expected. Substitution ofposition 1 resulted in an increase in recognition. TABLE III TILN LAU203 TILN LAU 132 Peptide Relative Relative Sequence [nM] activity [nM]activity AAGIGILTV 60 1 30 1 ALGIGILTV >1000 <0.6 >1000 <0.03AMGIGILTV >1000 <0.6 >1000 <0.03 LAGIGILTV 6 10 1.5 20 MAGIGILTV 8 7.52.5 12 EAAGIGILTV 12 5 3 10 ELAGIGILTV 2 30 0.05 600 EMAGIGILTV 2 300.05 600 EALGIGILTV >1000 <0.06 >1000 <0.03 EAMGIGILTV >1000 <0.06 >1000<0.03 YAAGIGILTV 5 20 1 30 FAAGIGILTV 1 60 0.05 600

[0050] The results obtained with CTLs are presented herein.Specifically, five independent HLA-A*0201 restricted Melan-A specificCTL clones were used, each of which is known to lyse melanoma targetcells.

[0051] The CTLs recognized SEQ ID NO: 2 with varying efficiency. WhenLeu was used to substitute Ala at position 1, four of the five clonesshowed enhanced recognition, while similar substitutions at position 2resulted in a loss of activity. Three of the five clones recognized SEQID NO: I more efficiently than SEQ ID NO: 2, but all recognized SEQ IDNO: 9 very efficiently, while recognition of SEQ ID NO: 10 resulted indecreased efficiency of recognition to differing degrees, and SEQ ID NO:11 resulted in reduced recognition for four of five. When SEQ ID NO: 12was tested, it was surprising that recognition improved, because TILrecognition decreased. With respect to SEQ ID NOS: 13 and 14, there wasreduced recognition by the CTLs.

[0052] It can be gathered from this that SEQ ID NOS: 7 and 9 were betterrecognized, consistently, than the other peptides tested, while otherpeptides w,ere recognized to different degrees. TABLE IV Recognition ofpeptide analogs by Melan-A specifice CTL clones Recog- nition M77.867.10 by done M77.80 1.13 Peptide Peptide Relative Peptide RelativePeptide Relative Peptide Relative Peptide Relative sequence [nM] 50%activity [nM] 50% activity [nM] 50% activity [nM] 50% activity [nM] 50%activity AAGIGLTV 15 1 50 1 300 1 300 1 4000 1 ALGIGLTV 90 0.16 >1000<0.015 >1000 <0.3 >1000 <0.3 >10000 <0.4 AMGIGLTV >1000 <0.015 >1000<0.015 >1000 <0.3 >1000 <0.3 >10000 <0.4 LAGIGILTV 0.08 187 1.5 33 150 20.03 10000 30 130 MAGIGLTV 0.6 .25 15 3 200 1.5 0.5 600 80 50 EAAGIGILTV0.15 100 4 12 0.06 5000 600 0.5 2000 2 EALGIGILTV 300 0.05 >1000 <0.01540 7.5 >1000 <0.3 >10000 <0.4 EAMGIGLTV 0.5 30 1 50 0.02 15000 5 60 5080 ELAGIGLTV 0.015 1000 0.5 100 0.015 20000 0.5 600 20 200 EMAGIGLTV 55036 >1000 <0.015 40 7.5 >1000 <0.3 >10000 <0.4 YAAGIGILTV 0.015 1000 351.4 >1000 <0.3 1000 0.3 >10000 <0.4 FAAGIGLTV 0.005 3000 7 7 >1000<0.3 >1000 <0.3 200 20

[0053] Additional experiments are depicted in FIG. 3 which showrecognition of various Melan-A peptide analogues presented by T2 cells,by TILN LAU203 and TILN LAU132. A 4-hour ⁵¹Cr assay was conducted at alymphocyte to target ration of 30:1.

[0054] The first panels of FIG. 3 (top and bottom) compare SEQ ID NOS:2, 7, 8, 5, and 6.

[0055] The second set of panels (top and bottom) compare SEQ ID NOS: 1,9, 10, 11, and 12.

[0056] The third set (top and bottom) compares SEQ ID NOS: 1, 13, 14,and 4.

[0057] The most important result obtained herein, however, was the factthat CTLS, induced with SEQ ID NO: 9 did recognize and lyse cellspresenting the endogenous peptide when SEQ ID NO: 9 was used.

EXAMPLE 6

[0058] Based upon the preceding data, the peptide of SEQ ID NO: 9 wasused for CTL induction studies.

[0059] In accordance with Valmori, et al, supra, peripheral bloodlymphocytes from HLA-A*0201 positive melanoma patients were purified bycentrifugation, and were enriched for CD3⁺ cells. The enrichedsubpopulation was then selected for CD8⁺ cells. The resultingsubpopulations routinely contained more than 90% CD8⁺ cells, and thesewere used in fuirther experiments.

[0060] The purified, CD8⁺ T cells were plated, at 1-2×10⁶ cells/well,together with 2×10⁶ stimulator cells, the preparation of which isdiscussed, infra. The effector and stimulator cells were combined in atotal of 2 ml of Iscove's medium which had been supplemented with 10%human serum, L-arginine (0.55 mM), L-asparagine (0.24 mM), andL-glutamine (1.5 mM), together with recombinant human IL-7 (10 ng/ml)and recombinant human IL-2 (10 U/ml).

[0061] To prepare the stimulator cells, 2×10⁶ autologous PBLs wereincubated for 2 hours, at 37° C., in serum free medium, with 20 μg/ml ofeach peptide and 3 μg/ml β2-microglobulin. The PBLs were then washed,irradiated, (3000 rads), and then adjusted to an appropriate volume,before being added to the CD8⁺ cell populations. On day 7, cells wererestimulated with peptide pulsed, autologous PBLs in complete medium,supplemented with 10 ng/ml of recombinant human IL-7, and 10 U/ml ofrecombinant human IL-2. There were weekly restimulations, using PBLswhich were peptide pulsed and irradiated. CTL activity was tested forthe first time after the second cycle (MC-2).

[0062] The results are shown in the following table and in FIG. 4. InFIG. 4, the source of CD8⁺ cells used was LAU203. CTL activity wasassayed seven days after the second (MC-2) restimulation. Results wereobtained using SEQ ID NOS: 1 and 2. These were used to permit comparisonto SEQ ID NO: 9.

[0063] Note that there was barely any activity with the parentalpeptides in sample LAU203, while SEQ ID NO: 9 elicited a strong CTLresponse. This activity was also cross reactive with SEQ ID NO: 1.

[0064] The results in the following table describe experiments using thesame peptides and using PBL from eight different HLA-A2 positivemelanoma patents, LAU203, LAU132, LAU145, LAU86, LAU50, LAU148, LAU161and LAU119. TABLE V Percentage specific lysis from cultures stimulatedwith peptide^(a)) SEQ ID NO: 2 SEQ ID NO: 1 SEQ ID NO: 9 tested onMelan-A 27-35 Melan-A 26-35 Melan-A 26-35 A27L Patient T2 + T2 + T2 +code E/T^(b)) T2 M10 Me260 Me290 T2 M10 Me290 Me260 T2 M10 Me290 Me260LAU 203 100  35^(c)) 29 7 17 37 41 15 6 52 83 18 81 30 29 11 10 0 17 237 1 26 96 4 75 10  3 6 2 0 9 17 0 0 17 73 1 62 LAU 132 100  9 12 1 0 1919 6 3 34 50 6 81 30  3 7 2 0 5 10 1 2 16 52 3 18 10  0 0 5 1 0 0 1 0 523 2 6 LAU 145 100 15 24 4 1 39 40 5 9 29 10 6 80 30  9 12 3 1 15 25 2 110 29 5 19 10  3 6 0 0 4 6 0 0 10 16 3 7 LAU 86 100 36 29 22 5 44 38 1410 35 45 24 15 30 17 15 9 5 20 26 6 0 24 23 10 4 10 16 5 2 0 10 10 1 014 9 1 0 LAU 50 100 21 26 7 5 18 20 5 5 19 26 6 20 30  7 16 4 5 8 13 1 010 18 3 8 10  7 7 0 4 0 4 1 0 3 12 0 0 LAU 148 100 31 39 13 4 46 45 9 034 39 9 4 30 19 8 5 4 20 26 1 2 19 27 9 3 10  3 6 1 0 14 14 6 0 13 13 10 LAU 161 100 24 22 6 1 23 31 3 1 25 38 4 23 30  3 8 6 1 16 12 3 0 15 232 13 10  2 0 5 0 9 7 2 0 5 11 3 4 LAU 119 100 31 27 5 12 23 31 1 4 18 465 45 30  7 13 1 1 17 23 3 4 13 39 4 23 10  4 0 0 0 9 12 1 0 7 17 2 16Clone 6 10  7 78 2 73 3  3 74 0 61 1  0 65 0 51 #Bold face type indicatesignificant specific CTL. When the differences in specific lysisobtained on T2 cells in presence or in absence of Melan-A 26-35 (1 μM)or Me290 and Me260 is equal or higher than 10%. A patient is consideredas responder when a significant specific lysis is detected in at leastone of the cultures.

EXAMPLE 7

[0065] As was pointed out, supra, the decapeptide of SEQ ID NO: 1 had ahigher efficiency of recognition than the nonamer of SEQ ID NO: 2.Experiments were carried out to determine if this was the result ofbetter binding of the peptide of SEQ ID NO: 1 to HLA-A^(·)0201molecules. These involved a functional peptide competition assay. Thistype of assay is described by Gaugler et al., J. Exp. Med. 174: 921(1994), incorporated by reference, but is described herein. In thisassay, HLA-A^(·)0201 expressing target cells (T2 cells) were labelledwith ⁵¹Cr and than incubated, for 15 minutes, with varyingconcentrations of peptides. A suboptimum concentration of mutant Ras5-14 peptide was added. This peptide has amino acid sequence: Lys LeuVal Val Val Gly Ala Val (SEQ ID NO: 20) Gly Val.

[0066] After 15 minutes a sample of CTL clone 7 RAS was added. This CTLclone had been obtained from the draining lymph node of an HLA-A^(·)0201human/B-microglobulin double transgenic mouse that had been injectedwith the peptide of SEQ ID NO: 20. The CTLs were added at a ratio of 5lymphocytes (10,000 cells/well):l target cell. The cells were incubatedat 37° C. for four hours, and then the assay was terminated. In additionto the peptide of SEQ ID NO: 1, those of SEQ ID NO: 2, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 3, SEQ ID NO: 4, and Glu Ala Asp Pro Thr GlyHis (SEQ ID NO: 21) Ser Tyr,

[0067] were tested. SEQ ID NO: 21 is a known peptide, derived fromMAGE-1, which is known to bind to HLA-A1 molecules and stimulate lysis.See U.S. Pat. No. 5,405,940, SEQ ID NO: 12, incorporated by reference.

[0068] When SEQ ID NO: 20 was used alone, the lysis percentage was 80%.Control lysis, with no peptide, was 4%.

[0069] The results indicated that SEQ ID NO: 1 showed five fold moreefficient binding than SEQ ID NO: 2. Both SEQ ID NOS: 3 and 4 bound withactivities comparable to SEQ ID NO: 2, while the control (SEQ ID NO:21), showed no binding. Both of SEQ ID NOS: 13 and 14 showedsignificantly improved binding as compared to SEQ ID NO: 2. Similarresults were obtained when a human CTL clone, specific for a complex ofHLA-A^(·)0201 and a different peptide, was used. The table which followspresents relative competitor activity as concentration of SEQ ID NO: 2required to inhibit control lysis by 50%, divided by concentration ofpeptide being tested to secure the same result: TABLE VI RelativeCompetitor Activity Peptide Exp1 Exp2 Exp3 SEQ ID NO: 2 1 1 1 SEQ ID NO:1 4 4 5 SEQ ID NO: 14 12 17 20 SEQ ID NO: 13 8 10 10 SEQ ID NO: 3 NotDone Not Done 2 SEQ ID NO: 4 Not Done Not Done 2

EXAMPLE 8

[0070] Relative HLA-A^(·)0201 peptide binding activity was thendetermined in another assay, based upon a flow cytometric assay. Inthese experiments, 2×10⁵ T2 cells were incubated with varyingconcentrations of the peptides of SEQ ID NO: 1, 2, 13, 14 or 4, for 16hours, at 23° C., in the presence of 2 ug/ml of human β₂ microglobulin.Cells were washed, at 4° C., and then stained with monoclonal antibodyBB7.2, labelled with FITC. This mAb is specific for a conformationdependent epitope on HLA-A2 molecules. Fluorescence index was thencalculated, by using the formula (mean fluorescence of sample—meanfluorescence of background)/(mean fluorescence of background). SeeNijman et al., Eur. J. Immunol. 23: 1215 (1993). Again, SEQ ID NO: 1showed highest efficiency of binding (about 10 fold) than SEQ ID NO: 2.SEQ ID NO: 4 showed relative binding activity about the same as SEQ IDNO: 2, while SEQ ID NOS: 13 and 14 had binding activities comparable toSEQ ID NO: 1.

EXAMPLE 9

[0071] In these experiments, efficiency of peptide recognition wasassessed with a panel of 13 CTL clones, all of which were specific forcomplexes of HLA-A^(·)0201 and SEQ ID NO: 2.

[0072] The same type of ⁵¹Cr release assay as is described in, e.g.,example 3, supra was carried out. Specifically, T2 cells were labelledwith ⁵¹Cr (the sodium salt was used), in Tris-Dulbecco buffer,supplemented with 2 mg/ml bovine serum albumin, and a 1:40 dilution ofW6/32 ascites as described by Gaugler et al, supra. This stabilizes theMHC molecule. The labelled cells were added to varying concentrations ofpeptides, and in varying amounts so as to create differingeffector:target ratios. Ten of the thirteen CTL clones tested recognizedthe decapeptide of SEQ ID NO: 1 more efficiently than the nonapeptide ofSEQ ID NO: 2, requiring concentrations of anywhere from 20 to more than1000 fold lower than the nonapeptide to achieve half-maximal lysis. Thethree remaining CTLs yielded titration curves which were similar. Noneof the CTLs recognized complexes of SEQ ID NO: 3 and HLA-A2. Inadditional experiments, one of the CTL clones was tested in IL-2 releaseassays, with SEQ ID NO: 1 again proving to be 10 fold more efficientthan SEQ ID NO: 2.

EXAMPLE 10

[0073] SEQ ID NOS: 1, 13, and 14 were then used to “dissect” the panelof the CTLs described supra. A recognition assay of the type describedin example 7, supra, was carried out using these peptides. Four of theCTLs recognized SEQ ID NOS: 1, 13 and 14 equally well. A fifth CTLrecognized SEQ ID NOS: 1 and 13, but not SEQ ID NO: 14. Two other CTLsrecognized SEQ ID NOS: 1 and 14, but not 13. One CTL only recognized SEQID NO: 1.

EXAMPLE 11

[0074] A set of experiments were then carried out to study the receptorsof the T cells described herein (“TCRs” hereafter), because it is knownthat different elements combine in the TCR repertoire, forming differentTCRs as a result.

[0075] To do this, total RNA of 10⁶ cells of each CTL clone tested wasextracted, following Chomczynski et al., Anal. Biochem. 162: 156 (1987).Then, reverse transcription using a poly(dT) primer was carried out,following the instructions in a commercially available product.Following this, aliquots of samples were PCR amplified, using a panel ofVα and Vβ probes, and Cα/Cβ specific oligonucleotides, in accordancewith Genevee, et al., Eur. J. Immunol. 22: 1261 (1992), incorporated byreference. Six different Vα segments were found, i.e., Vα2, 4, 6, 7, 14,and 21. One clone actually presented two in-frame Vα transcripts. Sevendifferent Vβ segments were found (two clones expressed Vβ13, twoexpressed Vβ14, and two expressed Vβ16. Vβ2, Vβ3, Vβ7.2 and Vβ8.2 wereeach expressed by one clone).

EXAMPLE 12

[0076] The determination of the contribution of single amino acid sidechains to the interaction between SEQ ID NO: 1 and HLA-A^(·)0201molecule, was studied, by testing single Ala substituted derivatives.That is to say, derivatives were prepared which were identical to SEQ IDNO: 1 but for a change at position 1, 4, 5, 6, 7, 8, 9, or 10 to Ala.

[0077] The peptides were prepared, following standard synthetic methods.Then, they were tested in a functional competition assay based upontheir ability to inhibit binding of a known HLA-A^(·)0201 bindingpeptide, i.e.

Tyr Met Asp Gly Thr Met Ser Gln Val (SEQ ID NO: 22),

[0078] derived from tyrosinase, and an HLA-A^(·)0201 restricted CTLclone, LAU 132/2. In brief, T2 cells were labelled with ⁵¹Cr in thepresence of monoclonal antibody W6/32. Both the cells and monoclonalantibody are described supra. Varying concentrations of competitorpeptide (50 ul volumes) were incubated with 50 ul of the ⁵¹Cr labelledT2 cells (This quantity gives 1000 cells/well) for 15 minutes, at roomtemperature. Then, 1 nM of the peptide of SEQ ID NO: 22 was added, whichis a suboptimal dose, together with 5000 CTLs, per well (a volume of 50ul). ⁵¹Cr release was measured after incubating for four hours at 37° C.The concentration of each competitor peptide required to inhibit ⁵¹Crrelease by 50% was determined. Comparison was facilitated by calculatingrelative competitor activity as the concentration of SEQ ID NO: 1 neededfor 50% inhibition, divided by the 50% inhibition value for the testpeptide.

[0079] It was found that substituting Ala for Glu at position 1 (SEQ IDNO: 15) resulted in a 5 fold increase in competitor activity.Substitution of Glu at position 4 or 6 by Ala resulted in decreasedactivity, of 20 and 10 fold, as did substitution of positions 7 and 10(about 25 fold, each time). Changes at positions 8 or 9, i.e. Glu AlaAla Gly Ile Gly Ile Ala Thr Val (SEQ ID NO: 23) and Glu Ala Ala Gly IleGly Ile Leu Ala Val (SEQ ID NO: 24)

[0080] did not result in significant changes in activity.

EXAMPLE 13

[0081] The stability of complexes formed by the single Ala substitutionsof SEQ ID NO: 1, discussed supra, and HLA-A^(·)0201 was then studied.Briefly, T2 cells were loaded with saturating concentrations (10 uM) ofthe analogs, and incubated overnight at room temperature with thepeptide and β2 microglobulin (3 ug/ml), in serum free medium. Excesspeptide was removed, and emetine (10⁻⁴M) was added to block proteinsynthesis. Cells were then incubated, for varying time periods, andaliquots were stained with fluorescent labelled anti HLA-A2 monoclonalantibody (BB7.2), to determine the amount of the molecules on thesurface. Since the peptide of SEQ ID NO: 17, supra, is known to formstable complexes with HLA-A^(·)0201 (van der Burg, J. Immunol. 156: 3308(1996)), this peptide was used as an internal standard. Complexstability was determined by calculating mean fluorescence with analog,subtracting background fluorescence and dividing by the same valuesobtained using SEQ ID NO: 17. Background fluorescence was the valueobtained using T2 cells treated similarly, but without peptide.

[0082] It was found that, over a period of 1-6 hours, complexes of SEQID NO: 1/HLA-A^(·)0201 were unstable, and dissociated in less than anhour. SEQ ID NO: 15 formed stable complexes over the same 6-hour period.All other derivatives tested formed complexes with low stability.

EXAMPLE 14

[0083] The derivatives described supra were then tested for theirrelative antigenic activity. In these experiments, two TILN populations,i.e., TILN LAU 132 and TILN LAU 203, preparation of which is described,supra, and a panel of ten different cytolytic T cell lines were tested.Of the ten CTLs, five were derived from infiltrating lymphocytes ortumor infiltrating lymph nodes, and five were from normal donorperipheral blood lymphocytes. All were known to be specific forcomplexes of HLA-A^(·)0201 and SEQ ID NO: 2; however, given the resultsdiscussed supra, showing superiority of SEQ ID NO: 1, this decapeptidewas used for comparison.

[0084] Antigen recognition was assessed in a ⁵¹Cr release assay. Target,T2 cells were labelled with ⁵¹Cr for one hour at 37° C., then washed,twice. The labelled target cells (1000 cell samples in 50 ul) were thenincubated with varying concentrations of peptides (in 50 ul volume), for15 minutes at room temperature, before adding effector cells (50 ul).When TILNs were the effector cells, these had been preincubated for atleast 20 minutes at 37° C., with unlabelled K562 cells (50,000cells/well), to eliminate non-specific lysis due to NK-like effectors.The ⁵¹Cr was measured in supernatant, harvested after four hours ofincubation at 37° C. Percent lysis was determined by subtractingspontaneously released ⁵¹Cr from ⁵¹Cr released with the tested, dividedby a figure obtained by subtracting spontaneous release from total ⁵¹Cr,and multiplying the resulting figure by 100. Titration was carried outover concentrations ranging from 10⁻⁵ to 10⁻¹³M. For quantitativecomparison, concentrations required for 50% maximal activity, normalizedagainst reference values for SEQ ID NO: 2 were determined.

[0085] SEQ ID NO: 15 was found to be recognized 20-60 fold better thanthe parental decapeptide of SEQ ID NO: 1, by the two TILN populations,in contrast to the other variants tested. With respect to the CTLs, 8 of10 of those tested recognized the peptide better than they recognizedSEQ ID NO: 1 or 2.

[0086] Additional differences were observed with respect to the CTLspecificity. Five of the ten CTLs tested recognized SEQ ID NO: 1 betterthan SEQ ID NO: 2. One of these five CTLs recognized SEQ ID NO: 1efficiently, and SEQ ID NO: 2 poorly. Three of the remaining five clonesrecognized SEQ ID NOS: 1 and 2 equally efficiently, and two recognizedSEQ ID NO: 2 better than SEQ ID NO: 1.

[0087] What this shows is a strong degree of diversity in finespecificity of tumor reactive CTLs.

EXAMPLE 15

[0088] A further set of experiments were then carried out to determineif diversity of antigenic specificity, as displayed by the CTL clones,supra, was a general characteristic of the T cell repertoire for theantigen SEQ ID NO: 1 or was attributable to different methodologies usedto derive the CTLs. To test this, the source of the specific T cells hadto be one which had not been subjected to antigen driven selection fromin vitro stimulation with peptides. The TILN population LAU 203,described supra, was used. As was shown in the prior examples, thepopulation exhibits relatively high CTL activity against Melan-AHLA-A^(·)0201 positive melanoma cells. CTL clones were derived from thispopulation, by limiting dilution cultures, in the presence ofirradiated, allogenic PBMCs, Epstein Barr Virus transformed Blymphocytes, phytohemagglutinin, and recombinant IL-2. Using standardprobability models, clones were derived from cultures having probableclonality of higher than 90%. These were then expanded, by plating 5×10³cells, every 3-4 weeks, into microtiter plates, together with irradiatedfeeder cells (5×10⁴ allogenic PBMCs, and 2.10⁴ EBV transformed B cells),with PHA and recombinant IL-2.

[0089] Two independent experiments were carried out, resulting in 130growing clones. When these were tested in a ⁵¹Cr release assay, of thetype described supra, 11 of these were found to recognize at least oneof SEQ ID NO: 1 and 2.

[0090] In order to determine fine specificity, the type of antigenrecognition assay described supra was carried out, using SEQ ID NOS: 1,2 and 15. It was found that four clones recognized SEQ ID NO: 1 betterthan SEQ ID NO: 2 (i.e., relative antigenic activity was at least 10fold greater), six clones recognized the two peptides equally well, andone recognized SEQ ID NO: 2 better than SEQ ID NO: 1. Nine CTLsrecognized SEQ ID NO: 15 better than SEQ ID NO: 1 and 2, with one of theclones actually recognizing SEQ ID NO: 15 at nanomolar concentrations,in contrast to micromolar concentrations for the peptides SEQ ID NO: 1and SEQ ID NO: 2.

EXAMPLE 16

[0091] The in vitro immunogenicity of the peptides of the invention wasdetermined. To do this, PBMCs from LAU 203 (1.0×10⁷ cells per test),were stimulated by adding 1 μM of peptide into the culture mediumcontaining the cells. The peptides tested were those defined by SEQ IDNOS: 1, 2, 9, 15 and 16. The peptide defined by SEQ ID NO: 17 was usedas a negative control.

[0092] Following the addition of the peptides, the cultures werestimulated weekly with autologous PBMCs which had been pulsed for 1 hourwith one of the peptides listed supra, at 37° C. In other words, aculture treated with SEQ ID NO: 1 was restimulated by having PBMCspulsed with SEQ ID NO: 1. The restimulating cells were washed,thoroughly, and irradiated prior to their use.

[0093] Seven days after stimulation, the cultures were monitored todetermine presence of CD8⁺ cells, which were reactive with tetramers ofHLA-A2 and SEQ ID NO: 9. This step was repeated a total of three times,over a 3-week period. To make the tetramers, it was first necessary toprepare constructs which would encode modified HLA-A^(·)0201 molecules.To do this, total RNA was extracted from HLA-A^(·)0201 positive cells,and HLA-A^(·)0201 was then cloned, using specific primers for themolecule, and reverse transcription polymerase chain reaction (RT-PCR).Altman et al., Science 274:94-96 (Oct. 4, 1996) incorporated byreference, was followed. Simultaneously, with the RT-PCR, the aminoterminal nucleotide sequence was altered to optimize protein expressionin the vector used. See Garboczi et al., Proc. Natl. Acad. Sci. USA89:3429 (1992) incorporated by reference. Once this was done, theextracellular coding portion of the molecule was amplified, again usingspecific primers. The resulting construct was recloned into a vectorwhich would produce a BirA biotinylation recognition site in frame atthe 3′-end of the HLA-A^(·)0201 heavy chain. The modified HLA-A^(·)0201and 32 microglobulin were overexpressed in separate E. coli cultures.The resulting inclusion bodies were purified and the HLA and β2microglobulin recombinant proteins were solubilized into urea, and thenrefolded in a refolding solution, at 4° C. to form complexes. (Therefolding solution contained 100 mM Tris, at pH 8.0, L-arginine, 400 mM,EDTA, 2 mM, reduced glutathione, 5 mM, oxidized glutathione, 0.5 mM,PMSF, 0.1 mM, HLA heavy chain and B2 microglobulin, 1 μM, and 10 μM ofthe peptide of interest). The refolding solution was concentrated to 7.5ml, using standard techniques. Then, refolding buffer was exchanged withBirA reaction buffer (Tris 100 mM, pH 7.5, NaCl 200 mM, Mg Cl₂ 5 mM,PMSF 100 μM, leupeptin 1 μM, and pepstatin 1 μM), the last three beingadded immediately before use.

[0094] The complexes were then biotinylated with biotin holoenzymesynthase (the BirA enzyme) by combining the refold mix containing theHLA-A2 complex with 50 μM enzyme, 100 mM biotin in 200 mM Tris, and 100mM adenosine triphosphate. The mixture was incubated overnight at roomtemperature. The biotinylated complexes were then purified, and combinedwith phycoerythrin-labeled streptavidin, to produce tetramericstructures. These were isolated, and reconstituted in small volumes, ata concentration of 1 mg/ml.

[0095] Peptides of SEQ ID NO: 9 were added to bind to the tetramers. Thetotal number of T cells positive for the tetramers relative to the totalnumber of CD8⁺ cells in each sample was determined. These results areshown in FIG. 5. The analogues were all found to have induced CD8⁺ cellsspecific for SEQ ID NO: 9.

EXAMPLE 17

[0096] Seven days after the third stimulation cycle, the cultures weretested in a ⁵¹Cr release assay to determine if they could lyse T2 cellsin the presence or absence of SEQ ID NO: 1. These assays were carriedout essentially as described in example 14, supra. The results arepresented in FIG. 6. It was found that stimulation with the analoguesactually resulted in more vigorous expansion of CD8⁺ cells reactive withthe tetramers described supra, than did stimulation with SEQ ID NO: 1 orSEQ ID NO: 2. Further, the Melan-A specific lysis correlated directlywith the percentage of the CD8⁺ cells which were also tetramer specific,suggesting comparable lytic capacity.

EXAMPLE 18

[0097] A further set of experiments were carried out to study theantigen specificity of CD8⁺, tetramer positive cells. To do this, theCD8⁺ tetramer positive cells were purified from each culture usingstandard flow cytometry sorting methods. The cells were then expanded invitro, using standard mitogen stimulation techniques. They were thentested for their lytic activity on T2 cells, following the method of theprevious example, in the presence or absence of the peptides used in thein vitro stimulation outlined supra, or SEQ ID NO: 1.

[0098] The results, presented in FIG. 7 show that each culture exhibiteda high level of specific lysis against both target cells pulsed with thetesting peptide and with SEQ ID NO: 1.

[0099]FIG. 7 also documents tumoricidal capacity of different cultures,by assessing this capacity against autologous Melan-A⁺ melanoma line Me290. See example 1, supra. High tumoricidal activity was observed, with50% maximal tumoricidal activity being observed at an effector/targetratio of3:1 for SEQ ID NO: 2, 7:1 for SEQ ID NO: 1, 4:1 for SEQ ID NO:15, 5:1 for SEQ ID NO: 9, and 15:1 for SEQ ID NO: 16.

EXAMPLE 19

[0100] Affinity of antigen recognition and relative antigenic activityof the different cell populations was then quantitated, using a standardCTL assay, along the lines of the assay of example 3, supra. Variousranges of peptide concentrations were used to develop titration curves,one of which is shown in FIG. 8. These data are summarized in Table VII,which follows: TABLE VIII Relative Potency of Melan-A Monospecific CTLLines After Tetramer-Guided Fluorescent Cytometry Sorting. Culturestimulated with SEQ ID NO: 2 1 15 9 16 (A) Peptide [nM] 50%  2 25 20 2535 50  1 1 2.5 3 3 15 15 0.04 0.04 0.15 0.08 0.15  9 0.001 0.15 0.030.03 0.3 16 0.001 0.015 0.01 0.003 0.03 (B) Relative antigenic activity: 2 1 1 1 1 1  1 25 8 8 12 3 15 625 500 166 437 333  9 25 × 10³ 133 8331666 166 16 25 × 10³ 1333 2500 16 × 10³ 1666

[0101] The affinity of the different lines for parental peptides wasvery similar, except for the line obtained after in vitro stimulationwith SEQ ID NO: 16. This line was found to recognize complexes ofHLA-A2/SEQ ID NO: 1 about 2 fold less efficiently, and complexes ofHLA-A2/SEQ ID NO: 1 5 to 15 fold less efficiently than other cell lines.

[0102] With respect to the second part of Table VIII it must be notedthat regardless of the peptide used to stimulate expansion, all of thecell lines recognized complexes containing SEQ ID NO: 1 better thancomplexes containing SEQ ID NO: 2. Peptide analogues were recognizedmore efficiently than parental sequences by all lines, notwithstandingdifferences in relative antigenicity for different lines. A preferenceof a cell line for an analogue did not always correlate with theanalogue used to generate the cell line.

[0103] The foregoing examples, as will be seen, describe the variousfeatures of the invention. These include peptides which bind to HLAmolecules, such as HLA-A2 molecules, exemplified by HLA-A*0201, whichmay also provoke proliferation of cytolytic T cells. These peptides, aswill be seen from the data herein, are nonapeptides or decapeptides. Aswith all peptides, the first amino acid is the amino terminus, and thelast one is the carboxy terminus. The peptides of the invention may bedecapeptides, which have a Val moiety at the C, or carboxy terminus.They may have at the amino terminus, Tyr or Phe when the second aminoacid is Ala. In another embodiment, the amino terminus is Glu followedby Ala, Leu or Met in the second and third position, and terminate withVal, wherein if position two is Ala, position three must be Met or Leu,and vice versa. The peptides having the amino acid sequences set forthin any of SEQ ID NOS: 5 and 8-14 are exemplary.

[0104] Also a part of the invention are isolated cytolytic T cell lineswhich are specific for complexes of these peptides and their MHC bindingpartner, i.e., an HLA molecule, such as an HLA-A2 molecule, HLA-A*0201being especially preferred.

[0105] The ability of these peptides to bind to HLA molecules makes themuseful as agents for determining presence of HLA-A2 positive cells, suchas HLA-A*0201 positive cells, by determining whether or not the peptidesbind to cells in a sample. This “ligand/receptor” type of reaction iswell known in the art, and various methodologies are available fordetermining it.

[0106] A further aspect of the invention are so-called “mini genes”which carry information necessary to direct synthesis of modifieddecapeptides via cells into which the mini genes are transfected. Minigenes can be designed which encode one or more antigenic peptides, andare then transferred to host cell genomes via transfection withplasmids, or via cloning into vaccinia or adenoviruses. See, e.g.,Zajac, et al., Int. J. Cancer 71: 496 (1997), incorporated by reference.

[0107] The peptides may be combined with peptides from other tumorrejection antigens to form ‘polytopes’. Exemplary peptides include thoselisted in U.S. patent application Ser. Nos. 08/672,351, 08/718,964, nowU.S. Pat. No. ______, Ser. No. 08/487,135 now U.S. Pat. No. ______ Ser.Nos. 08/530,569 and 08/880,963 all of which are incorporated byreference.

[0108] Additional peptides which can be used are those described in thefollowing references, all of which are incorporated by reference: U.S.Pat. Nos. 5,405,940; 5,487,974; 5,519,117; 5,530,096; 5,554,506;5,554,724; 5,558,995; 5,585,461; 5,589,334; 5,648,226; and 5,683,886;PCT International Publication Nos. 92/20356; 94/14459; 96/10577;96/21673; 97/10837; 97/26535; and 97/31017 as well as pending U.S.application Ser. No. 08/713,354.

[0109] Polytopes are groups of two or more potentially immunogenic orimmune stimulating peptides, which can be joined together in variousways, to determine if this type of molecule will stimulate and/orprovoke an immune response.

[0110] These peptides can be joined together directly, or via the use offlanking sequences. See Thompson et al. Proc. Natl. Acad. Sci. USA92(13): 5845-5849 (1995), teaching the direct linkage of relevantepitopic sequences. The use of polytopes as vaccines is well known. See,e.g., Gilbert et al., Nat. Biotechnol. 15(12): 1280-1284 (1997); Thomsonet al., supra; Thomson et al., J. Immunol. 157(2): 822-826,(1996); Tamet al., J. Exp. Med. 171(1): 299-306 (1990), all of which areincorporated by reference. The Tam reference in particular shows thatpolytopes, when used in a mouse model, are useful in generating bothantibody and protective immunity. Further, the reference shows that thepolytopes, when digested, yield peptides which can be and are presentedby MHCs. Tam shows this by showing recognition of individual epitopesprocessed from polytope ‘strings’ via CTLs. This approach can be used,e.g., in determining how many epitopes can be joined in a polytope andstill provoke recognition and also to determine the efficacy ofdifferent combinations of epitopes. Different combinations may be‘tailor-made’ for the patients expressing particular subsets of tumorrejection antigens. These polytopes can be introduced as polypeptidestructures, or via the use of nucleic acid delivery systems. Toelaborate, the art has many different ways available to introduce DNAencoding an individual epitope, or a polytope such as is discussedsupra. See, e.g., Allsopp et al., Eur. J. Immunol. 26(8); 1951-1959(1996), incorporated by reference. Adenovirus, pox-virus, Ty-virus likeparticles, plasmids, bacteria, etc., can be used. One can test thesesystems in mouse models to determine which system seems most appropriatefor a given, parallel situation in humans. They can also be tested inhuman clinical trials.

[0111] Also, a feature of the invention is the use of these peptides todetermine the presence of cytolytic T cells in a sample. It was shown,supra, that CTLs in a sample will react with peptide/MHC complexes.Hence, if one knows that CTLs are in a sample, HLA-A2 positive cells canbe “lysed” by adding the peptides of the invention to HLA-A2 positivecells, such as HLA-A*0201 positive cells, and then determining, e.g.,radioactive chromium release, TNF production, etc. or any other of themethods by which T cell activity is determined. Similarly, one candetermine whether or not specific tumor infiltrating lymphocytes(“TILs”) are present in a sample, by adding one of the claimed peptideswith HLA-A2 positive cells to a sample, and determining lysis of theHLA-A2 positive cells via, e.g., ⁵¹Cr release, TNF presence and soforth. In addition, CTL may be detected by ELISPOT analysis. See forexample Schmittel et al. (1997). J. Immunol. Methods 210: 167-174 andLalvani et al (1997). J. Exp. Med. 126: 859 or by FACS analysis offluorogenic tetramer complexes of MHC Class I/peptide (Dunbar et al(1998), Current Biology 8: 413-416. All are incorporated by reference.

[0112] Of course, the peptides may also be used to provoke production ofCTLs. As was shown, supra, CTL precursors develop into CTLs whenconfronted with appropriate complexes. By causing such a “confrontation”as it were, one may generate CTLs. This is useful in an in vivo context,as well as ex vivo, for generating such CTLs.

[0113] Other features of the invention will be clear to the skilledartisan, and need not be repeated here.

[0114] The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, it beingrecognized that various modifications are possible within the scope ofthe invention.

1 32 1 10 PRT Artificial sequence Peptide based on Melan-A peptide 1 GluAla Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 2 9 PRT Artificial SequencePeptide derived from Melan A 2 Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 310 PRT Artificial Sequence Peptide based on Melan A protein 3 Ala AlaGly Ile Gly Ile Leu Thr Val Ile 1 5 10 4 9 PRT Artificial SequencePeptide based on Melan A protein 4 Ile Leu Thr Val Ile Leu Gly Val Leu 15 5 9 PRT Artificial Sequence Derivative of SEQ ID NO 2 5 Ala Leu GlyIle Gly Ile Leu Thr Val 1 5 6 9 PRT Artificial Sequence Derivative ofSEQ ID NO 2 6 Ala Met Gly Ile Gly Ile Leu Thr Val 1 5 7 9 PRT ArtificialSequence Derivative of SEQ ID NO 2 7 Leu Ala Gly Ile Gly Ile Leu Thr Val1 5 8 9 PRT Artificial Sequence Derivative of SEQ ID NO 2 8 Met Ala GlyIle Gly Ile Leu Thr Val 1 5 9 10 PRT Artificial Sequence Derivative ofSEQ ID NO 1 9 Glu Leu Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 10 10 PRTArtificial sequence Derivative of SEQ ID NO 1 10 Glu Met Ala Gly Ile GlyIle Leu Thr Val 1 5 10 11 10 PRT Artificial sequence Derivative of SEQID NO 1 11 Glu Ala Leu Gly Ile Gly Ile Leu Thr Val 1 5 10 12 10 PRTArtificial Sequence Derivative of SEQ ID NO 1 12 Glu Ala Met Gly Ile GlyIle Leu Thr Val 1 5 10 13 10 PRT Artificial sequence Derivative of SEQID NO 1 13 Tyr Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 14 10 PRTArtificial Sequence Derivative of SEQ ID NO 1 14 Phe Ala Ala Gly Ile GlyIle Leu Thr Val 1 5 10 15 10 PRT Artificial sequence Derivative of SEQID NO 1 15 Ala Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 16 10 PRTArtificial Sequence Derivative of SEQ ID NO 1 16 Ala Leu Ala Gly Ile GlyIle Leu Thr Val 1 5 10 17 9 PRT H. influenzae Portion of Influenza Amatrix protein 17 Glu Ile Leu Gly Phe Val Phe Thr Leu 1 5 18 9 PRTArtificial Sequence Portion of Influenza A matrix protein 18 Gly Val AspPro Ile Gly His Leu Tyr 1 5 19 9 PRT Artificial Sequence Peptide fromprotein MAGE-3 19 Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 20 10 PRTArtificial Sequence Mutant Ras 5-14 Peptide 20 Lys Leu Val Val Val GlyAla Val Gly Val 1 5 10 21 9 PRT Artificial Sequence Peptide from MAGE-1Protein 21 Glu Ala Asp Pro Thr Gly His Ser Tyr 1 5 22 9 PRT ArtificialSequence Peptide derived from tyrosinase which binds HLA-A*0201 22 TyrMet Asp Gly Thr Met Ser Gln Val 1 5 23 10 PRT Artificial SequenceDerivative of SEQ ID NO 15 23 Glu Ala Ala Gly Ile Gly Ile Ala Thr Val 15 10 24 10 PRT Artificial Sequence Derivative of SEQ ID NO 15 24 Glu AlaAla Gly Ile Gly Ile Leu Ala Val 1 5 10 25 10 PRT Artificial SequenceDerivative of SEQ ID NO1 25 Glu Ala Ala Ala Ile Gly Ile Leu Thr Val 1 510 26 10 PRT Artificial Sequence Derivative of SEQ ID NO1 26 Glu Ala AlaGly Ala Gly Ile Leu Thr Val 1 5 10 27 10 PRT Artificial SequenceDerivative of SEQ ID NO1 27 Glu Ala Ala Gly Ile Ala Ile Leu Thr Val 1 510 28 10 PRT Artificial Sequence Derivative of SEQ ID NO1 28 Glu Ala AlaGly Ile Gly Ala Leu Thr Val 1 5 10 29 10 PRT Artificial SequenceDerivative of SEQ ID NO1 29 Glu Ala Ala Gly Ile Gly Ile Ala Thr Val 1 510 30 10 PRT Artificial Sequence Derivative of SEQ ID NO 1 30 Glu AlaAla Gly Ile Gly Ile Leu Ala Val 1 5 10 31 10 PRT Artificial SequenceMUTAGEN 1. 3...9 Dervative of SEQ ID NO1. Amino acid 1 is Ala, Tyr orPhe; amino acids 3-9 can be any amino acid 31 Xaa Ala Xaa Xaa Xaa XaaXaa Xaa Xaa Val 1 5 10 32 10 PRT Artificial Sequence MUTAGEN 2...9Derivative of SEQ ID NO1. Amino acids 4-9 are any amino acid. 32 Glu XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Val 1 5 10

1. An isolated peptide which binds to an HLA-A2 molecule, said peptideconsisting of ten amino acids, wherein the carboxy terminal amino acidis Val, the amino terminal amino acid is Ala, Tyr or Phe, and the secondamino acid is Ala.
 2. An isolated peptide which binds to an HLA-A2molecule and consists of ten amino acids, said isolated peptide havingVal at its carboxy terminus, Glu at its amino terminus, and the secondand third amino acids from the N-terminus are Ala, Leu, or Met, with theproviso that when the second amino acid is Ala, the third amino acidmust be Leu or Met, and when the third amino acid is Ala, the secondamino acid must be Leu or Met.
 3. The isolated peptide of claim 1, whichhas an, amino acid sequence selected from the group consisting of SEQ IDNO: 13, SEQ ID NO: 14, and SEQ ID NO:
 15. 4. The isolated peptide ofclaim 2, which has an amino acid sequence selected from the groupconsisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:12.
 5. An isolated cytolytic T cell line which specifically recognizes acomplex of the isolated peptide of claim 1 and an HLA-A2 molecule.
 6. Anisolated cytolytic T cell line which specifically recognizes a complexof the isolated peptides of claim 2 and an HLA-A2 molecule.
 7. A methodfor provoking proliferation of cytolytic T cells, comprising contactinga sample containing cytolytic T cells precursors with a complex of theisolated peptide of claim 1 and HLA-A2 molecules to provokeproliferation of any cytolytic T cell precursors specific to saidcomplex into cytolytic T cells.
 8. A method for provoking proliferationof cytolytic T cells, comprising contacting a sample containingcytolytic T cells precursors with a complex of the isolated peptide ofclaim 2 and HLA-A2 molecules to provoke proliferation of any cytolytic Tcell precursors specific to said complex into cytolytic T cells. 9.Isolated nonapeptide consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, and SEQ ID NO:
 8. 10. A method for determining presence of tumorinfiltrating lymphocytes (TILs) in a tumor sample, comprising admixingsaid tumor sample with a sample of HLA-A2 positive cells and theisolated peptide of claim 1, and determining lysis of said HLA-A2positive cells as a determination of TILs in said sample.
 11. A methodfor determining presence of tumor infiltrating lymphocytes (TILs) in atumor sample, comprising admixing said tumor sample with a sample ofHLA-A2 positive cells and the isolated peptide of claim 2, anddetermining lysis of said HLA-A2 positive cells as a determination ofTILs in said sample.
 12. A method for determining presence of tumorinfiltrating lymphocytes (TILs) in a tumor sample, comprising admixingsaid tumor sample with a sample of HLA-A2 positive cells and theisolated peptide of claim 9, and determining lysis of said HLA-A2positive cells as a determination of TILs in said sample.
 13. Isolateddecapeptide consisting of the amino acid sequence set forth in SEQ IDNO: 3, SEQ ID NO: 23, or SEQ ID NO:
 24. 14. Isolated decapeptide whichhas an amino acid sequence selected from the group consisting of SEQ IDNO: 13, SEQ ID NO: 14, and SEQ ID NO:
 16. 15. A method for provokingproliferation of a cytolytic T lymphocyte which is reactive with a cellthat presents, on its surface, a complex of an HLA-A2 molecule and apeptide which forms a non-covalent complex with said HLA-A2 moleculecomprising contacting a sample containing said cytolytic T lymphocytewith a complex of an HLA-A2 molecule and a nonamer or decamer, whereinthe carboxy terminal amino acid for said nonamer or decamer is Val, theamino terminal amino acid for said nonamer or decamer is Ala, Glu, Tyr,or Phe, and the second amino acid of said nonamer or decamer is Ala orLeu, so as to stimulate proliferation of cytolytic T cells which reactwith complexes of HLA-A2 and said nonamer or decamer, and at least oneother complex of HLA-A2 and a second, different nonamer or decamer. 16.The method of claim 15, wherein said nonamer or decamer has Ala or Gluas its N-terminal amino acid.
 17. The method of claim 15, wherein saidmonomer is the nonamer having the amino acid sequence of SEQ ID NO: 2.18. The method of claim 15, wherein said decamer is the decamer havingthe amino acid sequence of SEQ ID NO: 1, 9, 15, or 16.